Research Methods Chemocoding as an identification tool where morphological- and DNA-based methods fall short: Inga as a case study Marıa-Jose Endara1,2, Phyllis D. Coley1,3, Natasha L. Wiggins4, Dale L. Forrister1, Gordon C. Younkin1, James A. Nicholls5, R. Toby Pennington6, Kyle G. Dexter6,7, Catherine A. Kidner5,6, Graham N. Stone5 and Thomas A. Kursar1,3 1Department of Biology, University of Utah, Salt Lake City, UT 84112-0840, USA; 2Centro de Investigacion de la Biodiversidad y Cambio Climatico (BioCamb) e Ingenierıa en Biodiversidad y Recursos Geneticos, Facultad de Ciencias de Medio Ambiente, Universidad Tecnologica Indoamerica, Quito EC170103, Ecuador; 3Smithsonian Tropical Research Institute, Box 0843-03092, Balboa, Ancon, Republic of Panama; 4School of Biological Sciences, University of Tasmania, Sandy Bay, TAS 7001, Australia; 5Ashworth Labs, Institute of Evolutionary Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, EH9 3JY, UK; 6Royal Botanic Garden Edinburgh, Edinburgh, EH3 5LR, UK; 7School of GeoSciences, University of Edinburgh, Edinburgh, EH9 3FF, UK Summary Author for correspondence: The need for species identification and taxonomic discovery has led to the development of Marıa-Jose Endara innovative technologies for large-scale plant identification. DNA barcoding has been useful, Tel: +1 801 581 7086 but fails to distinguish among many species in species-rich plant genera, particularly in tropical Email: [email protected] regions. Here, we show that chemical fingerprinting, or ‘chemocoding’, has great potential Received: 4 August 2017 for plant identification in challenging tropical biomes. Accepted: 4 January 2018 Using untargeted metabolomics in combination with multivariate analysis, we constructed species-level fingerprints, which we define as chemocoding. We evaluated the utility of New Phytologist (2018) chemocoding with species that were defined morphologically and subject to next-generation doi: 10.1111/nph.15020 DNA sequencing in the diverse and recently radiated neotropical genus Inga (Leguminosae), both at single study sites and across broad geographic scales. Key words: chemocoding, Inga, Our results show that chemocoding is a robust method for distinguishing morphologically metabolomics, species identification, similar species at a single site and for identifying widespread species across continental-scale tropical forests. ranges. Given that species are the fundamental unit of analysis for conservation and biodiversity research, the development of accurate identification methods is essential. We suggest that chemocoding will be a valuable additional source of data for a quick identification of plants, especially for groups where other methods fall short. Introduction (Gemeinholzer et al., 2006; Hollingsworth et al., 2009, 2011; Chen et al., 2010; Kress et al., 2010; China Plant BOL Group Cataloguing the world’s plant diversity has been a challenge for et al., 2011). Here we present evidence that chemical fingerprint- centuries, and because of accelerated anthropogenic extinctions, ing or ‘chemocoding’ can be another tool for species identifica- the rapid documentation of biodiversity is more critical than ever tion in confusing and/or closely related tree species in (Mace, 2004; Valentini et al., 2009; Vernooy et al., 2010; Cris- challenging, hyperdiverse biomes. tescu, 2014). This is particularly true in tropical rainforests, No single identification method is without drawbacks. Mor- where the high diversity and co-occurrence of morphologically phological methods are often labor-intensive, rely upon taxo- and ecologically similar congeneric species have presented signifi- nomic expertise and are the most prone to subjective errors, cant challenges for identification (Gonzalez et al., 2009; Dexter particularly where phenotypic plasticity and cryptic taxa are et al., 2010; Kress et al., 2015; Liu et al., 2015). Plant species have prevalent (de Carvalho et al., 2005; Costion et al., 2011). typically been identified by botanical experts based on morpho- Although DNA barcoding is rapid and straightforward, it can fail logical characteristics, or more recently by sequencing several to distinguish closely related plant species because of insufficient chloroplast DNA regions or the internal transcribed spacer of sequence divergence in standard barcode markers (Kress et al., nuclear ribosomal DNA (ITS), referred to as ‘DNA barcoding’ 2009; Dexter et al., 2010; Liu et al., 2015) and may lead to faulty Ó 2018 The Authors New Phytologist (2018) 1 New Phytologist Ó 2018 New Phytologist Trust www.newphytologist.com New 2 Research Phytologist identifications in genera where species are of recent origin 2012). Inga is one of the most abundant and diverse Neotropical (Razafimandimbison et al., 2004; Naciri & Linder, 2015; Pen- genera in lowland forest communities (Valencia et al., 1994; ter nington & Lavin, 2015). These limitations are often exacerbated Steege et al., 2013), is widely distributed and has undergone in highly diverse tropical systems, as the proportion of species recent, rapid diversification (Richardson et al., 2001). For Inga, belonging to young, species-rich genera is high (Richardson et al., genetic and morphological differentiation of closely related 2001). For example, in a subtropical Chinese forest, for the 44% species is low and the identification of a species can therefore be of species belonging to genera with more than two species, > 50% difficult. shared barcoding sequences and could not be distinguished (Liu We propose the use of small, defense-related chemical markers et al., 2015). This led to an overall species resolution of only 67- characterized via untargeted metabolomics in combination with %. A similar problem is encountered in New World tropical rain- multivariate analysis for the construction of a phytochemical, forests, where DNA barcoding cannot reliably discriminate species-level fingerprint, which we define as chemocoding. We species within ecologically important, species-rich genera such as evaluate the units defined by chemocoding with those defined Inga, Ficus and Piper (Gonzalez et al., 2009; Kress et al., 2009). morphologically in a recent taxonomic monograph (Pennington, While standard DNA barcoding for problematic groups can be 1997) as well as with those defined using next-generation DNA improved by adding data for additional loci, recently diverged sequencing data of many hundreds of nuclear genes (Nicholls species will always be hard to distinguish using sequence data and et al., 2015; our unpublished data). no standardized marker sets for such extended DNA barcoding exist. Materials and Methods Many studies assessing diversity in surveys or plots for conser- vation or basic science rely on identifying all individuals in a plot Study sites to species level, including the large majority of individuals that are without flowers or fruits (Dexter et al., 2010). Plots in the Samples were collected at five sites that include a wide range of tropics represent a daunting task, and are still faced with prob- soils but very similar climates throughout the Amazon and lematic identifications despite extremely well-trained botanists. A Panama (Fig. 1). Barro Colorado Island is a field station adminis- related problem is the difficulty of achieving uniform species tered by the Smithsonian Research Tropical Institute located in identifications across multiple sites. For example, in a recent, extensive analysis of the identifications for eight genera, the three genera with the highest error rates were Andira and Tachigali (c. 50%) and Inga (c. 40%; Baker et al., 2017). And yet another sub- stantial challenge is correlating the identities to species level for saplings, small trees and adult trees, because ontogenetic changes in leaf morphology may be considerable and juveniles do not bear flowers or fruits. For example, a consortium to understand forest dynamics has established 63 plots around the world where all woody plants > 1 cm diameter at breast height (DBH) are mapped and identified, the majority of which are juveniles (www.forestgeo.si.edu). Thus, the issues and errors associated with morphological species identifications of thousands of trees in the tropical forests are serious. Given that species are a fundamental unit of analysis for con- servation, for quantifying biodiversity, and for understanding ecological and evolutionary processes, the development of accu- rate methods for identifying them is essential. In this paper, we suggest that chemical fingerprinting (here termed chemocoding) can provide an additional identification tool for species identifi- cation in a species-rich tropical tree genus, particularly for mor- phologically confusing or cryptic species. We examine its utility both for distinguishing species within a single site, and for char- acterizing within-species variation over wider geographic scales. Moreover, chemocoding may be inexpensive enough to allow for every individual tree to be tested. We test the potential of chemocoding for species identification within Inga Mill. (Leguminosae, Mimosoideae) because species in this genus are difficult to distinguish morphologically and Fig. 1 Study sites: (1) Barro Colorado, Panama, (2) Nouragues, French show insufficient variation in barcoding sequences (Richardson Guiana, (3) Tiputini, Ecuador, (4) Los Amigos, Peru, and (5) KM41 near et al., 2001; Kress et al., 2009; Dexter et al., 2010; Dick & Webb, Manaus, Brazil. New Phytologist (2018) Ó 2018 The Authors www.newphytologist.com New Phytologist Ó 2018